PROJECT SUMMARY The persistence of HIV within long-lived reservoirs results in the need for life-long antiretroviral therapy (ART). The accurate quantification of these reservoirs is challenging due to the relatively small number of latently infected cells within an individual. The optimal assay for HIV reservoir quantification should be high-throughput, sensitive, cost-efficient, and would ideally be able to quantify the frequency of HIV-infected cells. Current assays to measure the size of these reservoirs are hindered by a number of drawbacks, including the inability to determine infected cell numbers, the need for large sample volumes, and the cost and labor-intensive nature of certain assays. A single-cell assay that can quantify the number of infected cells with active HIV transcription would provide an unprecedented level of clarity on the state of the HIV reservoir, the effects of latency- reversing agents, and would have important implications for mechanistic and basic pathogenesis studies as well. We have recently validated a single-cell platform for detecting the frequency of HIV-expressing cells. Using this assay, we propose to evaluate the effectiveness of a range of latency-reversing agents for HIV reactivation and to explore a possible underlying mechanism behind their differential effects.